Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

Newton, Jared M.; Hanoteau, Aurélie; Sikora, Andrew G.

doi:10.3791/57685

Cited by 5 publications

(1 citation statement)

References 14 publications

(16 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The filtered cells are stained with fluorescent antibodies such as anti‐CD4, and anti‐CD8, to investigate the frequency of CD4+ helper and CD8+ cytotoxic T cells with flow cytometry. [ 148 ] To further understand T cell distribution in tumor, tumor tissues are thinly sliced and stained with fluorescent antibodies. [ 149 ] Confocal imaging is used concurrently with tissue histology to understand the spatial distribution of tumor‐infiltrating lymphocytes in tumor tissue and extracellular matrix.…”

Section: Biological Assays To Assess Transfection Performancementioning

confidence: 99%

Materials for Improving Immune Cell Transfection

et al. 2021

View full text Add to dashboard Cite

recognize and bind to tumor-associated antigens such as CD19 and epidermal growth factor receptor (EGFR) before releasing cytotoxic granules and effector cytokines to destroy cancer cells. [3] CAR-T therapy has shown remarkable success against CD19 expressing B cell hematological malignancies and, thus far, five products (Kymriah from Novartis; Yescarta and Tecartus from Kite Pharma/Gilead Sciences; and Breyanzi and Abecma from Bristol-Myers Squibb) have been approved by the US Food and Drug Administration (FDA) for clinical use. [4,5] Currently, there are at least 500 clinical trials registered on ClinicalTrials.gov database using engineered immune cells to treat various cancers including that of the brain, lungs, and skin.Despite the clinical success of CAR-T cells against B cell leukemia, CAR-T therapy still face tremendous challenges in manufacturing, safety, and affordability before it can become a viable clinical option. [6] One of the biggest technical difficulties is to transfect sensitive, primary T cells whereby biomolecules like oligonucleotides and proteins have to be delivered intracellularly and into the nuclei of cells. FDA-approved gold standard viruses and bulk electroporation suffer from low transfection efficiency while also perturbing the critical biological attributes of cells such as proliferation, metabolism, and gene expression. [7] This increases the time and costs for cell expansion, and without an efficient and cost-friendly transfection technology, the price (between USD 0.4-0.5 million per patient) for FDA-approved CAR treatments (Kymriah and Yescarta) will remain unaffordable and untimely. [8] The limitations in conventional transfection techniques have motivated the development of micro-and nanoplatforms such as microfluidics, nanoparticles, and high-aspect-ratio nanostructures to improve immune cell viability and throughput during transfection.Herein, we first provide an overview of CAR-T cell manufacturing, with emphasis on the science of transfection and limitations of traditional technology using viruses and bulk electroporation. There will also be discussion of other cell-based cancer immunotherapy using other types of promising immune cell types. Next, we describe emerging transfection platforms and companies that have been established to overcome gaps in CAR-T transfection. We then provide a list of assays constituting the polyfunctionalities of immune cells that we believe will help the field better assess the robustness and suitability of their transfection methods. Finally, we end off with existing challenges in CAR-T transfection and how overcoming these challenges can significantly enhance the clinical impact of CAR-T therapy.Chimeric antigen receptor T cell (CAR-T) therapy holds great promise for preventing and treating deadly diseases such as cancer. However, it remains challenging to transfect and engineer primary immune cells for clinical cell manufacturing. Conventional tools using viral vectors and bulk electroporation suffer from low efficiency while posing risks ...

show abstract

Section: Biological Assays To Assess Transfection Performancementioning

confidence: 99%

Materials for Improving Immune Cell Transfection

et al. 2021

View full text Add to dashboard Cite

show abstract

Sustained Intratumoral Administration of Agonist CD40 Antibody Overcomes Immunosuppressive Tumor Microenvironment in Pancreatic Cancer

et al. 2023

View full text Add to dashboard Cite

Agonist CD40 monoclonal antibodies (mAb) is a promising immunotherapeutic agent for cold‐to‐hot tumor immune microenvironment (TIME) conversion. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal cancer known as an immune desert, and therefore urgently needs more effective treatment. Conventional systemic treatment fails to effectively penetrate the characteristic dense tumor stroma. Here, it is shown that sustained low‐dose intratumoral delivery of CD40 mAb via the nanofluidic drug‐eluting seed (NDES) can modulate the TIME to reduce tumor burden in murine models. NDES achieves tumor reduction at a fourfold lower dosage than systemic treatment while avoiding treatment‐related adverse events. Further, abscopal responses are shown where intratumoral treatment yields growth inhibition in distant untreated tumors. Overall, the NDES is presented as a viable approach to penetrate the PDAC immune barrier in a minimally invasive and effective manner, for the overarching goal of transforming treatment.

show abstract

Glutathione peroxidase 2 is a metabolic driver of the tumor immune microenvironment and immune checkpoint inhibitor response

Ahmed

Veeramachaneni

Deng

et al. 2022

J Immunother Cancer

View full text Add to dashboard Cite

BackgroundThe existence of immunologically ‘cold tumors’ frequently found across a wide spectrum of tumor types represents a significant challenge for cancer immunotherapy. Cold tumors have poor baseline pan-leukocyte infiltration, including a low prevalence of cytotoxic lymphocytes, and not surprisingly respond unfavorably to immune checkpoint (IC) inhibitors. We hypothesized that cold tumors harbor a mechanism of immune escape upstream and independent of ICs that may be driven by tumor biology rather than differences in mutational neoantigen burden.MethodsUsing a bioinformatic approach to analyze TCGA (The Cancer Genome Atlas) RNA sequencing data we identified genes upregulated in cold versus hot tumors across four different smoking-related cancers, including squamous carcinomas from the oral cavity (OCSCC) and lung (LUSC), and adenocarcinomas of the bladder (BLCA) and lung (LUAD). Biological significance of the gene most robustly associated with a cold tumor phenotype across all four tumor types, glutathione peroxidase 2 (GPX2), was further evaluated using a combination of in silico analyses and functional genomic experiments performed both in vitro and in in vivo with preclinical models of oral cancer.ResultsElevated RNA expression of five metabolic enzymes including GPX2, aldo-keto reductase family 1 members AKR1C1, AKR1C3, and cytochrome monoxygenases (CP4F11 and CYP4F3) co-occurred in cold tumors across all four smoking-related cancers. These genes have all been linked to negative regulation of arachidonic acid metabolism—a well-established inflammatory pathway—and are also known downstream targets of the redox sensitive Nrf2 transcription factor pathway. In OCSCC, LUSC, and LUAD, GPX2 expression was highly correlated with Nrf2 activation signatures, also elevated in cold tumors. In BLCA, however, GPX2 correlated more strongly than Nrf2 signatures with decreased infiltration of multiple leukocyte subtypes. GPX2 inversely correlated with expression of multiple pro- inflammatory cytokines/chemokines and NF-kB activation in cell lines and knockdown of GPX2 led to increased secretion of prostaglandin E2 (PGE2) and interleukin-6. Conversely, GPX2 overexpression led to reduced PGE2 production in a murine OCSCC model (MOC1). GPX2 overexpressing MOC1 tumors had a more suppressive tumor immune microenvironment and responded less favorably to anti-cytotoxic T-lymphocytes-associated protein 4 IC therapy in mice.ConclusionGPX2 overexpression represents a novel potentially targetable effector of immune escape in cold tumors.

show abstract

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

Cited by 5 publications

References 14 publications

Materials for Improving Immune Cell Transfection

Materials for Improving Immune Cell Transfection

Sustained Intratumoral Administration of Agonist CD40 Antibody Overcomes Immunosuppressive Tumor Microenvironment in Pancreatic Cancer

Glutathione peroxidase 2 is a metabolic driver of the tumor immune microenvironment and immune checkpoint inhibitor response

Contact Info

Product

Resources

About